Nucleotide-specific recognition sequences for designer TAL effectors

Abstract

The invention relates to methods of altering expression of a genomic locus of interest or specifically targeting a genomic locus of interest in an animal cell, which may involve contacting the genomic locus with a non-naturally occurring or engineered composition that includes a deoxyribonucleic acid (DNA) binding polypeptide having a N-terminal capping region, a DNA binding domain comprising at least five or more Transcription activator-like effector (TALE) monomers and at least one or more half-monomers specifically ordered to target the genomic locus of interest, and a C-terminal capping region, wherein the polypeptide includes at least one or more effector domains, and wherein the polypeptide is encoded by and translated from a codon optimized nucleic acid molecule so that the polypeptide preferentially binds to the DNA of the genomic locus.

We claim: 
     
       1. A deoxyribonucleic acid (DNA) binding polypeptide comprising:
 (a) a N-terminal capping region or fragment thereof, 
 (b) a DNA binding domain comprising at least five Transcription activator-like effector (TALE) monomers and at least one half-monomer specifically ordered to target a genomic locus of interest, and 
 (c) a C-terminal capping region or fragment thereof, 
 wherein the polypeptide includes at least one M.SssI DNA methyl transferase (DNMT) domain, and 
 wherein the polypeptide is capable of binding to DNA of the genomic locus of interest in a mammalian cell and regulating expression of the genomic locus by site-specific cytosine methylation at the genomic locus. 
 
     
     
       2. The polypeptide of  claim 1 , wherein the DNA binding domain comprises (X 1-11 -X 12 X 13 -X 14-33 or 34 or 35 ) z ,
 wherein X 1-11  is a chain of 11 contiguous amino acids, 
 wherein X 12 X 13  is a repeat variable diresidue (RVD), 
 wherein X 14-33 or 34 or 35  is a chain of 21, 22 or 23 contiguous amino acids, 
 wherein z is at least 5 to 40, and 
 wherein at least one RVD is selected from the group consisting of NI, HD, NG, NN, NH, NQ, SS, SN, NK, HH, HI, HG, ND, YG, HN, NV, NS, HA, S*, N*, H*, NA, and NC, wherein (*) means that the amino acid at X 13  is absent. 
 
     
     
       3. The polypeptide of  claim 2 , wherein
 z is at least 10 to 26, or 
 at least one of X 1-11  is a sequence of 11 contiguous amino acids set forth as amino acids 1-11 in a sequence of X 1-11 -X 14-34  or X 1-11 -X 14-35  of SEQ ID Nos. 110, 111, 114, 116, 127, 185, 204, 257, 297, 338, 339 or 340 or 
 at least one of X 14-34  or X 14-35  is a sequence of 21 or 22 contiguous amino acids set forth as amino acids 12-32 or 12-33 in a sequence X 1-11 -X 14-34  or X 1-11 -X 14-35  of SEQ ID Nos. 110, 111, 114, 116, 127, 185, 204, 257, 297, 338, 339 or 340. 
 
     
     
       4. The polypeptide of  claim 1 , wherein the DNA binding domain comprises (X 1-11 -X 12 X 13 -X 14-33 or 34 or 35 ) z ,
 wherein X 1-11  is a chain of 11 contiguous amino acids, 
 wherein X 12 X 13  is a repeat variable diresidue (RVD), 
 wherein X 14-33 or 34 or 35  is a chain of 21, 22 or 23 contiguous amino acids, 
 wherein z is at least 5 to 40, and 
 wherein the at least one RVD is selected from the group consisting of (a) HH, NH, NK, NQ, SS for recognition of guanine (G); (b) HG for recognition of thymine (T); (c) RD, SD for recognition of cytosine (C); (d) NV for recognition of A or G; and (e) H*, HA, N*, NA, NC, NS, S* for recognition of A or T or G or C, wherein (*) means that the amino acid at X 13  is absent. 
 
     
     
       5. The polypeptide of  claim 4 , wherein the RVD for the recognition of G is NH. 
     
     
       6. The polypeptide of  claim 1 , wherein
 the N-terminal capping region or fragment thereof comprises 147 contiguous amino acids of a wild type N-terminal capping region, or 
 the C-terminal capping region or fragment thereof comprises 68 contiguous amino acids of a wild type C-terminal capping region, or 
 the N-terminal capping region or fragment thereof comprises 136 contiguous amino acids of a wild type N-terminal capping region and the C-terminal capping region or fragment thereof comprises 183 contiguous amino acids of a wild type C-terminal capping region. 
 
     
     
       7. A pharmaceutical composition comprising the polypeptide of  claim 1  and optionally one or more pharmaceutically acceptable excipients. 
     
     
       8. A non-naturally occurring or engineered composition comprising a nucleic acid molecule encoding a deoxyribonucleic acid (DNA) binding polypeptide, wherein the DNA binding polypeptide comprises:
 (a) a N-terminal capping region or fragment thereof, 
 (b) a DNA binding domain comprising at least five Transcription activator-like effector (TALE) monomers and at least one half-monomer specifically ordered to target a genomic locus of interest, and 
 (c) a C-terminal capping region or fragment thereof, 
 wherein the polypeptide includes at least one M.SssI DNA methyl transferase (DNMT) domain, and 
 wherein the polypeptide is capable of binding to DNA of the genomic locus of interest in a mammalian cell and regulating expression of the genomic locus by site-specific cytosine methylation at the genomic locus. 
 
     
     
       9. The composition of  claim 8 , further comprising an expression vector, wherein the nucleic acid molecule is cloned into the expression vector. 
     
     
       10. A method of selectively targeting a genomic locus of interest comprising a coding or a regulatory sequence susceptible to being affected by an effector domain in a mammalian cell, comprising contacting the genomic locus with a deoxyribonucleic acid (DNA) binding polypeptide comprising:
 (a) a N-terminal capping region or fragment thereof, 
 (b) a DNA binding domain comprising at least five Transcription activator-like effector (TALE) monomers and at least one half-monomer specifically ordered to target the genomic locus of interest, and 
 (c) a C-terminal capping region or fragment thereof, 
 wherein the polypeptide includes at least one M.SssI DNA methyl transferase (DNMT) domain, and 
 wherein the polypeptide is capable of binding to DNA of the genomic locus of interest in the mammalian cell and regulating expression of the genomic locus by cytosine methylation. 
 
     
     
       11. The method of  claim 10 , wherein the DNA binding domain comprises (X 1-11 -X 12 X 13 -X 14-33 or 34 or 35 )z,
 wherein X 1-11  is a chain of 11 contiguous amino acids, 
 wherein X 12 X 13  is a repeat variable diresidue (RVD), 
 wherein X 14-33 or 34 or 35  is a chain of 21, 22 or 23 contiguous amino acids, 
 wherein z is at least 5 to 40, and 
 wherein at least one RVD is selected from the group consisting of NI, HD, NG, NN, NH, NQ, SS, SN, NK, HH, HI, HG, ND, YG, HN, NV, NS, HA, S*, N*, H*, NA, and NC, wherein (*) means that the amino acid at X 13  is absent. 
 
     
     
       12. The method of  claim 11 , wherein
 z is at least 10 to 26, or 
 at least one of X 1-11  is a sequence of 11 contiguous amino acids set forth as amino acids 1-11 in a sequence of X 1-11 -X 14-34  or X 1-11 -X 14-35  of SEQ ID Nos. 110, 111, 114, 116, 127, 185, 204, 257, 297, 338, 339 or 340 or 
 at least one of X 14-34  or X 14-35  is a sequence of 21 or 22 contiguous amino acids set forth as amino acids 12-32 or 12-33 in a sequence X 1-11 -X 14-34  or X 1-11 -X 14-35  of SEQ ID Nos. 110, 111, 114, 116, 127, 185, 204, 257, 297, 338, 339 or 340. 
 
     
     
       13. The method of  claim 10 , wherein the DNA binding domain comprises (X 1-11 -X 12 X 13 -X 14-33 or 34 or 35 )z,
 wherein X 1-11  is a chain of 11 contiguous amino acids, 
 wherein X 12 X 13  is a repeat variable diresidue (RVD), 
 wherein X 14-33 or 34 or 35  is a chain of 21, 22 or 23 contiguous amino acids, 
 wherein z is at least 5 to 40, and 
 wherein the at least one RVD is selected from the group consisting of (a) HH, NH, NK, NQ, SS for recognition of guanine (G); (b) HG for recognition of thymine (T); (c) RD, SD for recognition of cytosine (C); (d) NV for recognition of A or G; and (e) H*, HA, N*, NA, NC, NS, S* for recognition of A or T or G or C, wherein (*) means that the amino acid at X 13  is absent. 
 
     
     
       14. The method of  claim 13 , wherein the RVD for the recognition of G is NH. 
     
     
       15. The method of  claim 10 , wherein
 the N-terminal capping region or fragment thereof comprises 147 contiguous amino acids of a wild type N-terminal capping region, or 
 the C-terminal capping region or fragment thereof comprises 68 contiguous amino acids of a wild type C-terminal capping region, or 
 the N-terminal capping region or fragment thereof comprises 136 contiguous amino acids of a wild type N-terminal capping region and the C-terminal capping region or fragment thereof comprises 183 contiguous amino acids of a wild type C-terminal capping region. 
 
     
     
       16. The method of  claim 10  wherein the method further comprises delivery of a nucleic acid molecule encoding the polypeptide to the mammalian cell. 
     
     
       17. A codon optimized nucleic acid molecule encoding a deoxyribonucleic acid (DNA) binding polypeptide, wherein the DNA binding polypeptide comprises:
 (a) a N-terminal capping region or fragment thereof, 
 (b) a DNA binding domain comprising at least five Transcription activator-like effector (TALE) monomers and at least one half-monomer specifically ordered to target a genomic locus of interest, and 
 (c) a C-terminal capping region or fragment thereof, 
 wherein the polypeptide includes at least one M.SssI DNA methyl transferase (DNMT) domain, and 
 wherein the polypeptide is capable of binding to DNA of the genomic locus of interest in a mammalian cell and regulating expression of the genomic locus by site-specific cytosine methylation at the genomic locus.